Circadian rhythm of hyperoxidized peroxiredoxin II is determined by hemoglobin autoxidation and the 20S proteasome in red blood cells

Abstract

The catalytic cysteine of the typical 2-Cys Prx subfamily of peroxiredoxins is occasionally hyperoxidized to cysteine sulfinic acid during the peroxidase catalytic cycle. Sulfinic Prx (Prx-SO 2 H) is reduced back to the active form of the enzyme by sulfiredoxin. The abundance of Prx-SO 2 H was recently shown to oscillate with a period of ∼24 h in human red blood cells (RBCs). We have now investigated the molecular mechanism and physiological relevance of such oscillation in mouse RBCs. Poisoning of RBCs with CO abolished Prx-SO 2 H formation, implicating H 2 O 2 produced from hemoglobin autoxidation in Prx hyperoxidation. RBCs express the closely related PrxI and PrxII isoforms, and analysis of RBCs deficient in either isoform identified PrxII as the hyperoxidized Prx in these cells. Unexpectedly, RBCs from sulfiredoxin-deficient mice also exhibited circadian oscillation of Prx-SO 2 H. Analysis of the effects of protease inhibitors together with the observation that the purified 20S proteasome degraded PrxII-SO 2 H selectively over nonhyperoxidized PrxII suggested that the 20S proteasome is responsible for the decay phase of PrxII-SO 2 H oscillation. About 1% of total PrxII undergoes daily oscillation, resulting in a gradual loss of PrxII during the life span of RBCs. PrxII-SO 2 H was detected in cytosolic and ghost membrane fractions of RBCs, and the amount of membrane-bound PrxII-SO 2 H oscillated in a phase opposite to that of total PrxII-SO 2 H. Our results suggest that membrane association of PrxII-SO 2 H is a tightly controlled process and might play a role in the tuning of RBC function to environmental changes.

Interplay between cellular redox oscillations and circadian clocks

“…Rhythmic overoxidation of PRDXs can be interpreted as a 'memory' of the flux going through its catalytic system and would therefore reflect underlying changes in the redox environment on the circadian time scale, rather than necessarily being a timekeeping mechanism in its own right. Consistent with this idea, mouse red blood cells from sulphiredoxin mutant animals still display rhythmic PRDX oxidation and the daily decline in PRDX overoxidation seems to be mainly due to proteasomal degradation [8].…”

“…Rhythmic overoxidation of PRDXs can be interpreted as a 'memory' of the flux going through its catalytic system and would therefore reflect underlying changes in the redox environment on the circadian time scale, rather than necessarily being a timekeeping mechanism in its own right. Consistent with this idea, mouse red blood cells from sulphiredoxin mutant animals still display rhythmic PRDX oxidation and the daily decline in PRDX overoxidation seems to be mainly due to proteasomal degradation [8].Importantly, the phylogenetic conservation of PRDX rhythms was subsequently extended to include mouse, insects, worms, bacteria and even Archaea, suggesting that circadian redox rhythms may be a common feature of all aerobic living organisms [7,44]. Critically, such rhythms are independent of previously identified transcriptional clocks, because mutants lacking circadian components maintain redox oscillations.…”

Enzymatic control of cysteinyl thiol switches in proteins

“…A crucial aspect for future studies on Prx-dependent redox regulation will be the identification of similar complementary patterns in Prx substrates and (potential) signal transducers. The physiological relevance of such interactions cannot be overemphasized considering that Prx are at the center of a plethora of redox-regulated processes and have excellent hydroperoxide sensor properties as outlined below (Delaunay et al, 2002;Jang et al, 2004;Brigelius-Flohé and Flohé, 2011;Edgar et al, 2012;Jarvis et al, 2012;Kil et al, 2012;Cho et al, 2014).…”

A 50-Year Personal Journey: Location, Gene Expression, and Circadian Rhythms

“…As mammalian red blood cells lack nuclei and therefore transcription, aspects of this very important hypothesis are likely to be correct. However, very specialized conditions were necessary to repeat the in vitro peroxiredoxin cycling, explaining perhaps why there was no peroxiredoxin cycling observed in red blood cells taken directly from animals in this study (Cho et al 2014). Importantly, no laboratory has independently replicated the experiments showing the cycling of hyperoxidized peroxiredoxin in flies and mice bearing arrhythmic mutations in key clock proteins (Edgar et al 2012).…”

“…A set of crucial experiments shows that these rhythms are independent of transcriptional rhythms in human red blood cells as well as in fly and mouse systems Edgar et al 2012). Importantly, key aspects of the red blood cell experiments were independently replicated (Cho et al 2014). As mammalian red blood cells lack nuclei and therefore transcription, aspects of this very important hypothesis are likely to be correct.…”